Understanding the effects of ethanol on neural stem cells is critical to unraveling the etiological knots of deficits associated with fetal alcohol spectrum disorder (FASD). FASD is a compelling problem because it is a major cause of developmental mental dysfunction affecting ~2% of all live births (indeed, it is the chief cause of mental retardation in the USA), and it provides insights into the etiology of other (often co-morbid) mental health disorders (e.g., attention deficit hyperactivity disorder and autism). The composition and size of the brain are established by the proliferation of neural stem cells and by the numbers and lineage of the derivatives. Improper numbers or balance of neuronal subpopulations can underlie mental dysfunction. Thus, we will test the hypothesis that ethanol affects the cycling behavior and fates of cells generated in the developing central nervous system. Cerebral cortex is composed of two types of neurons: excitatory projection neurons (PNs) and inhibitory local circuit neurons (LCNs). These derive prenatally from two distinct proliferative regions. PNs come from the dorsal telencephalon which comprises two zones: the ventricular zone (VZ) and its derivative, the subventricular zone (SZ). Most cortical LCNs are generated in the ventral telencephalon, in the medial ganglionic eminence (MGE). The fates of VZ/SZ and MGE cells are defined in a two-step process. (1) It is decided whether the cells remain in the cycling population and (2) the phenotype (e.g., the type of neuron) is defined. During the previous period of support, we showed that transforming growth factor (TGF) 21 is a key regulator of neural stem cell proliferation. The present project will explore the effects of ethanol on dynamics of cell proliferation and on the definition of cell fate. Specific Aims 1 and 2 will use an in vivo model to determine the effects of ethanol on the cycling activity (cell cycle kinetics and exit) and on the expression/activation of TGF2 receptors by neural stem cells in the cortical proliferative zones. During their development, neural stem cells express a homeobox gene product(s), Pax6 and/or Tbr2. These proteins define the transition from (Pax6+) stem cells in the VZ to an (Tbr2+) intermediate progenitor cell stage. This transition is critical for the development of superficial cortex (e.g., the origin of callosal projections) which is a target of prenatal exposure to ethanol. Specific Aim 3 will use two types of cultures (organotypic slices that retain in vivo-like organization and lines of neural stem cells harvested from the VZ/SZ or MGE) to explore the effects of ethanol on TGF21-regulated cell proliferation and fate decisions. In addition, we will identify genes that are up- and down-regulated and silenced (methylated) by ethanol and/or TGF21. In Specific Aim 4, neural stem cells will be transplanted (homotopically or heterotopically) to determine the effects of ethanol and/or TGF21 on genetic and environmental contributions to determining cycling behavior and to defining cell fate. In concert, the novel Aims will use three complementary models to gain critical insight into mechanisms defining cell proliferation and fate and the effects of ethanol on these critical factors. PUBLIC HEALTH RELEVANCE: Fetal alcohol spectrum disorder affects an estimated 2% of all live births in the United States. One common target of alcohol toxicity is proliferating cells, particularly neural stem cells that give rise to the brain. The present study will explore a mechanism by which alcohol-induced defects result - from changes (a) in the fates of proliferating neural stem cells and (b) in their response to a key regulator of that proliferation, transforming growth factor.